Download Immunization Against Luteinizing Hormone

Immunization Against Luteinizing
Hormone-Releasing Hormone Fusion Proteins
Does Not Decrease Prostate Cancer in the
Transgenic Adenocarcinoma Mouse
Prostate Model
RICHARD E. HILL,* DAVID M. DE AVILA,*,† KEVIN P. BERTRAND,‡ NORMAN M. GREENBERG,§
, ,1
AND JERRY J. REEVES* †
*Department of Animal Sciences, †Center for Reproductive Biology, and ‡School of Molecular
Biosciences, Washington State University, Pullman, Washington 99164; and §Department of Cell
Biology, Baylor College of Medicine, Houston, Texas 77030
This study was undertaken to test the effect of immunization
against luteinizing hormone-releasing hormone (LHRH) fusion
proteins on the development and progression of prostate cancer in the transgenic adenocarcinoma mouse prostate (TRAMP)
model. Two LHRH fusion proteins, ovalbumin with seven LHRH
peptides (OV-LHRH-7), and thioredoxin with seven LHRH peptides (TH-LHRH-7) were used in a cocktail vaccine. Two groups
of male TRAMP mice were immunized with the cocktail. Primary
immunizations were at either 4 or 8 weeks of age. LHRH immunized mice (n = 19) were compared with castrated (n = 19) and
intact mice (n = 18) for testosterone concentration, tumor
weight, and lifespan. Immunization against LHRH in the TRAMP
mice resulted in significant production of antibodies to LHRH
compared with surgically castrated and intact control mice.
Testicular weight was significantly reduced in the LHRH immunized groups compared with intact control mice. Serum testosterone was reduced (P < 0.05) in the immunized mice compared
with intact control mice and was not different from that of castrated mice (P > 0.05). Tumor weight was variable and inconsistent throughout all treatment groups. Lifespan was not increased by immunization against LHRH or castration. Intact
control mice (lived the longest (227 ± 11 days), whereas immunized mice lived 206 ± 11 days and castrated mice lived 213 ± 13
days. Tumors from immunized TRAMP mice appeared more aggressive than tumors of castrated and intact mice, as demonstrated by 35% expression of gross lung tumors in the immunized mice whereas none were observed in the castrated or
intact TRAMP mice. Prostate cancer is initially dependent upon
androgens for growth and development, but cells have the ability to escape androgen dependence and progress to an androgen independent state, which was evident in this study. The
1
To whom requests for reprints should be addressed at the Department of Animal
Sciences, 220 ASLB, Washington State University, Pullman, WA 99164–6353.
Received August 8, 2002.
Accepted February 10, 2003.
1535-3702/03/2287-0818$15.00
Copyright © 2003 by the Society for Experimental Biology and Medicine
818
TRAMP mouse model immunized against LHRH may have utility
in future studies and treatments of the androgen independent
prostate cancer. Exp Biol Med 228:818–822, 2003
Key words: TRAMP mouse; LHRH vaccine; prostate and lung cancer
H
uggins and Hodges (1) first discovered androgen
deprivation to be an effective treatment for prostate
cancer. As a result, many hormonal therapies, new
drugs, and surgical procedures have been developed for
reducing androgens to treat prostate cancer. Because of the
variability associated with the onset and progression of
prostate cancer in humans, models to study have been developed through the use of transgenics. The transgenic adenocarcinoma mouse prostate (TRAMP) model mouse (2)
was engineered using the prostate specific probasin (PB)
promoter coupled with the simian virus (SV)-40 Large T
antigen. Transgene expression occurs with sexual maturity
and results in progressive forms of prostate cancer that are
pathologically and histologically similar to human prostate
cancer. Characterization of this model has shown the onset
cancer is androgen-dependent prostatic intraepithelial neoplasia. This cancer progresses to androgen independence
and eventually metastasizes (3, 4).
The present study used two LHRH fusion proteins in
combination to immunize TRAMP mice. The LHRH fusion
proteins have either ovalbumin (OV) or thioredoxin (TH) as
carriers (5, 6). These two LHRH fusion proteins have been
shown to decrease testicular and accessory sex gland function and to reduce circulating testosterone and LH concentrations in male mice (6). The concept of immunological
castration and reduction of circulating androgens to treat
prostate cancer has been previously described by Ladd et al.
(7) and Simms et al. (8).
Several research groups have used the TRAMP model
for both treatment and chemoprevention studies (9–11). Im-
IMMUNIZATION DOES NOT DECREASE PROSTATE CANCER
munization against LHRH as a strategy for preventing prostate cancer has yet to be documented in the TRAMP mouse.
Previous studies have characterized the effect of androgen
ablation in the TRAMP model (4, 9, 10). In these studies it
was shown that early castration effectively decreases overall
tumor burden, but progression to androgen independence
was observed and was not ultimately delayed. Our hypothesis is that the LHRH immuno-castrated TRAMP mouse
will live longer than the castrated TRAMP mouse because
neutralization of LHRH in circulation will be beneficial in
decreasing androgen-independent prostate cancer. Fekete et
al. (12), Emons et al. (13), and Jungwirth et al. (14) have
reported that LHRH antagonists block LHRH receptors at
the tissue receptor site, which implicates LHRH in prostate
cancer progression. This study was designed to evaluate the
LHRH antibody response, testosterone concentration, and
lifespan of the male TRAMP mouse after LHRH immunological castration compared with surgically castrated
TRAMP mice and nontreated intact TRAMP mice.
Materials and Methods
Transgenic Animals. Male and female TRAMP
mice heterozygous for the PB-Tag transgene were maintained in a pure C57BL/6 colony. All animals were handled
according to the highest standards in accordance with the
NIH Guide for the Care and Use of Laboratory Animals
(Washington State University, LARC# 3080, 2995).
Both male and female C57BL/6 mice were mated with
FVB mice to produce C57BL/6 × FVB F1 offspring. Male
offspring were screened for the transgene using mouse tail
DNA isolation (15). Tail snips were boiled for 2 hr in 250
␮l of alkaline lysis (25 mM NaOH, 0.2mM EDTA, pH 12)
solution. Samples were cooled to 4°C and 250 ␮L of neutralizing reagent (40 mM Tris-HCl, pH 5) was added. Polymerase chain reaction (PCR) was performed using the
mouse ␤-casein (BCAS) gene as a marker and the Probasin-T antigen (PB-Tag) gene as the identifier. Three microliters each of the forward, the reverse BCAS primer, the Tag
forward, and the PB reverse primer were added to 3 ␮l of
the template DNA (tail sample). Fourteen microliters of
PCR Premix (Taq polymerase, dNTP, MgCl, sense and antisense primers; Amplicon Express©, Pullman, WA) was
added to the primer DNA solution and run through 30
cycles in a thermocycler. Product was then separated and
visualized using ethidium bromide on a 1.0% agarose gel.
LHRH Vaccine. The ovalbumin-LHRH-7 and thioredoxin-LHRH-7 fusion proteins were produced and isolated
as previously described (5, 6). All mice were vaccinated
with the same batch of purified proteins. Given in equimolar
quantities, a final concentration of 0.4 mM of each LHRH
protein was suspended in a total 100-␮l urea+adjuvant solution for immunization (5, 6). Modified Freund’s complete
adjuvant with Mycobacterium butyricum instead of Mycobaterium tuberculosis was used for the initial subcutaneous
vaccination and modified Freund’s incomplete adjuvant was
used for the two booster immunizations.
Experimental Design. Sixty positive TRAMP male
mice were identified from C57BL/6 (TRAMP) × FVB F1
offspring using PCR screening. The 60 mice were randomly
assigned into three treatment groups: LHRH immunized,
surgically castrated, and intact controls. Mice receiving the
LHRH vaccine were separated into two groups. The first
group (n ⳱ 10) received their primary injection at 4 weeks
of age with corresponding boosters at 6 and 8 weeks. The
second group (n ⳱ 10) received their primary immunization
at 8 weeks of age with corresponding boosters at 10 and 12
weeks. Two groups of 10 mice were castrated at either 6
weeks or 10 weeks, corresponding to the first booster immunization in each immunized group. Twenty mice were
left intact and untreated.
Blood Collection. Mice were maintained and bled
approximately once every 4 weeks after initial immunization until time of death or euthanasia. Mice were bled via
the saphenous vein as previously described (16). Briefly,
mice were placed in a clear holding tube. The hair was
trimmed on the rear leg to expose the saphenous vein. The
vein was swabbed with alcohol and then sterile lubricant
was placed around the area of the saphenous vein, which
was pricked using a sterile hypodermic needle. Fifty microliters of whole blood was collected in a heparinized capillary tube. The 50 ␮l of blood was diluted in 50 ␮l of
phosphate-buffered saline (PBS) and frozen until assayed
for LHRH antibodies. Mice were either found dead or determined to be close to death as indicated by prior to natural
death, as indicated by a roughened hair coat, lethargy, and
discomfort and were euthanized using carbon dioxide gas
followed by exsanguination. Final blood collection was performed via cardiac puncture and blood samples were processed as described above until assayed for LHRH antibodies and testosterone.
Necropsy and Histology. Mice living past the average lifespan (35 weeks) of a TRAMP mouse were genotyped after time of death. This revealed three negative
TRAMP mice, which were removed from the data, all others proved positive. At time of euthanization, internal organs of the animals were exposed by an incision in the
abdominal cavity. Internal organs, primarily urogenital organs, were visually examined for gross tumors, metastases,
and other abnormalities. Gross abnormalities were documented and recorded. Prostate tumors were removed and
weighed. Testes were then isolated and weighed. Sections
of prostate tumors and metastases were placed in Bouin’s
fixative. Tumors, seminal vesicles, lymph nodes, kidney,
liver, and lung samples were also taken and fixed from
representative mice. Samples were removed from the fixative after 1 hr and placed in 70% ethanol until sectioned and
stained.
Antibody Titers and Testosterone Assay. Iodination of LHRH was performed by placing an Iodobead® (Pierce) in 1 ml of 0.05M PBS (pH 7.5) for 2 to 5
min. In a separate tube, 5 ␮g of LHRH was added to 100 ␮l
of 0.05 M PBS and 1.0 ␮Ci (10 ␮l) I125 was added to the
IMMUNIZATION DOES NOT DECREASE PROSTATE CANCER
819
tube. The Iodo-bead® was dropped into the I125 solution
and reacted for 5 min. One milliliter of trifluoroacetic acid
(TFA) was added to the tube. The 125I-LHRH was separated
using reverse phase HPLC on a nucleosil C18 column (Phenomenex. Torrance, CA). Acetylnitrile 0.10% TFA and
0.12% TFA in H2O were used as buffers.
Antibody titers were measured using the supernatant of
centrifuged whole blood diluted 1:1000 in EDTA-PBS. One
hundred ␮L of 1:1000 serum was diluted in 300 ␮l of PBS
gel. Thirty thousand cpm of iodinated LHRH plus normal
mouse serum (1:400) was added to each tube. Samples were
incubated for 48 hr at 4°C followed by the addition of 100
␮l of sheep anti-mouse ␥-globulin (1:20) in PBS as the
second antibody. After incubation, samples were centrifuged at 3000g for 30 min, decanted, and pellets were
counted in a gamma counter. Percent binding of 125I-LHRH
was quantified as previously described (17). Testosterone
concentrations were measured by a radioimmunoassay kit
(DSL-4100) (Diagnostic Systems Laboratories, Inc.).
Statistical Analysis. Statistical analysis was performed using SAS statistical package. Proc GLM was used
to perform analysis of variance. LSD was used to determine
the difference between means for lifespan, testosterone concentration, testis weight, and tumor weight. Proc CORR was
used to determine correlation between antibody titers, testosterone concentrations and lifespan. ␹2 analysis was used
to determine difference in tumor occurrence associated with
organs. Significance was given at P < 0.05.
testosterone concentrations of both the 4 weeks and 8 weeks
treatment groups (Fig. 2A). Mean testis weight was reduced
(P < .05) in mice immunized at 4 weeks (157 ± 34 mg) and
8 weeks (210 ± 35 mg) compared with intact controls (340
± 10 mg; Fig. 2B). There was a tendency for lower testis
weight in the 4 weeks immunized mice (157 ± 34 mg)
compared to 8 weeks immunized mice (210 ± 35 mg) but
this difference was not significant (Fig. 2B).
The tumors of the TRAMP mice were heterogeneous in
size throughout the treatment groups, and there was no significant difference between tumor weight between any of
the treatment groups (Fig. 2C). Gross examination of tumors at necropsy revealed a difference in severity of metastatic tumors related to treatment. Gross lung tumors were
expressed in 35% (6/17) of the LHRH immunized mice and
0% (0/15) of the castrated and intact mice (0/16; Table I). In
some immunized mice, the kidney tumor was incorporated
into the primary prostate tumor of the TRAMP mouse, with
lymph nodes also fused to the primary prostate tumor. Of
the tumors examined histologically, only two mice from the
intact control TRAMP showed moderately differentiated tumors, all others were undifferentiated sheets of malignant
cells.
The overall lifespan of mice vaccinated at 4 weeks and
mice vaccinated at 8 weeks were not significantly different
from both groups of castrated mice. Although not statistically significant, the average lifespan of control mice tended
to be longer than either the castrated or immunized mice.
Results
Discussion
Immunization against two LHRH fusion proteins produced LHRH antibodies in all immunized mice. The neutralization of LHRH resulted in regression of the testes and
decreased testosterone concentrations similar to that of surgically castrated TRAMP mice. LHRH antibody titers were
higher in the 4 weeks LHRH immunized group when compared with the 8 weeks LHRH immunized group (Fig. 1).
However, there was no significant difference between the
Because the TRAMP mouse uses an androgen responsive transgene, it is a logical hypothesis that removal of
androgens will inhibit the development of androgen dependent prostate cancer in this mouse. In the present study,
mice were surgically castrated or immunized against LHRH
and allowed to live until death resulting from the prostate
cancer. Immunization against the two recombinant LHRH
fusion proteins produced LHRH antibodies in all treated
Figure 1. Percent binding of 125I-LHRH over time
in LHRH immunized, castrated, and intact control
TRAMP mice. Arrows represent primary immunizations at either 4 or 8 weeks and corresponding
boosters.
820
IMMUNIZATION DOES NOT DECREASE PROSTATE CANCER
Table I. Gross Necropsy Observations and
Anatomical Location of Tumors Identified in LHRH
Immunized, Surgically Castrated, and Intact Control
TRAMP Mice
Number of gross tumors observed
Treatment
n
Immunized
17
Castrated
15
Intact control 16
Prostate
Lymph Kidney Liver Lung
tumor
12
12
13
15
11
9
8
5
5
5
3
7
6a
0b
0b
Numbers in the columns with different superscripts represent significant differences (P < .01).
Figure 2. A, Average testosterone concentrations at time of death in
immunized, surgically castrated, and intact control TRAMP mice. B,
Average testis weights at time of death in immunized and intact
control TRAMP mice. C, Average tumor weights in immunized, surgically castrated, and intact control TRAMP mice. D, Average
lifespans of immunized, surgically castrated, and intact control
TRAMP mice. Numbers with different superscripts represents significant difference (P < 0.05).
TRAMP mice. Previous studies have suggested that using
two carriers may overcome carrier induced immune suppression (6, 17). LHRH antibody production led to a decrease in serum testosterone comparable to surgically castrated TRAMP mice and a significant decrease in testis size
compared to intact TRAMP mice. Androgen ablation has
been used as a strategy to reduce prostate carcinogenesis in
the TRAMP mouse. Androgen ablation has been successful
in inhibiting androgen dependent prostate cancer, which
was shown by a delay to the onset of a palpable tumor, but
mice were examined and killed before 20 weeks of age and
were not allowed to live out their lifespan (9, 10). In the
present study mice were allowed to live their entire lifespan.
Lifespan was estimated by death or by a physical appearance of lethargy, discomfort and roughened hair coat at
which time the individual mouse was euthanized. The androgen receptor is a key target in understanding the initiation and progression of prostate cancer. In the TRAMP
mouse, it plays a major role in transformation through its
stimulation of the probasin promoter (2). Recently, amplification of and mutations in the androgen receptor have
been associated with androgen-independent prostate cancer
in the TRAMP mouse and human prostate cancer (18–20).
Androgen ablation appears to result in production of androgen receptor variants that differ in sensitivity to androgens,
with some possibly having increased activity in the absence
of androgens.
The LHRH hormone has been implicated in androgenindependent prostate cancer by the presence of LHRH receptors found on tumors (12). LHRH antagonists and agonists have been shown to bind LHRH receptors in the prostate and exhibit a direct inhibitory effect on androgen
independent cancer cells (13, 14, 21, 22). Immunization
against LHRH in the present study does not allow LHRH to
bind to the receptors and therefore should nullify any effects
of LHRH on cancer cells, much like LHRH antagonists.
Contrary to previous studies using LHRH analogues to slow
cancer growth, immunization against LHRH does not result
in slowed growth of cancer in the TRAMP mouse. In this
study the tumors appeared to be more severe and invasive
than tumors of both the surgically castrated or intact control
TRAMP mice. It is possible that the kinetics of antagonist
binding results in the antiproliferative effect caused by the
administration of LHRH antagonists, and this effect cannot
occur with the neutralization of LHRH. It is also possible
that ample testosterone concentrations remained after immunization against LHRH, which stimulated the androgen
receptor variants produced as a result of the decreased testosterone concentrations. This coupled with adrenal androgen stimulation possibly produced the more aggressive, and
IMMUNIZATION DOES NOT DECREASE PROSTATE CANCER
821
invasive androgen independent tumors in the immunized
treatment groups.
In the present study, TRAMP mice that underwent androgen reduction via immunization against LHRH had statistically similar lifespans to the surgically castrated and
intact TRAMP mice. The present study was not designed to
evaluate androgen-dependent cancer alone but the sum effects of the dependent and independent prostate cancer together as measured by lifespan. Our hypothesis was that a
vaccine against LHRH would help combat prostate cancer
by 1) decreasing circulating testosterone thus slowing down
androgen dependent prostate cancer; and 2) the LHRH antibodies would decrease endogenous LHRH molecules from
stimulating androgen independent prostate cancer. Although the vaccine did produce LHRH antibodies, which
decreased both testes size and circulating testosterone, treatment was ineffective in extending lifespan. The removal of
LHRH from the system by immunization gave an unexpected increase in metastasis in the lung compared to the
intact and surgically castrated male. The TRAMP mouse
model immunized against LHRH may have utility in future
studies and treatments of the androgen independent prostate
cancer metastasizing to organs outside the local prostate.
1. Huggins C, Hodges CV. Studies in prostatic cancer II. The effect of
castration on advanced cancer of the prostate gland. Arch Surg
43:209–223, 1941.
2. Greenberg NM, Demayo FJ, Finegold MJ, Medina D, Tilley WD,
Aspinall JO, Cunha GR, Donjacour AA, Matusik RJ, Rosen JM. Prostate cancer in a transgenic mouse. Proc Natl Acad Sci USA 92:3439–
3443, 1995.
3. Gingrich J, Barrios R, Morton R, Boyce B, De Mayo F, Fingold M,
Angelopoulou R, Rosen J, Greenburg N. Metastatic prostate cancer in
a transgenic mouse. Cancer Res 56:4096–4102, 1996.
4. Gingrich J, Barrios R, Kattan M, Nahm H, Finegold M, Greenburg N.
Androgen independent prostate cancer progression in the TRAMP
model. Cancer Res 57:4687–4691, 1997.
5. Zhang Y, Rozell TG, de Avila DM, Bertrand KP, Reeves JJ. Development of recombinant ovalbumin-LHRH as a potential sterilization
vaccine. Vaccine 17:2185–2191, 1999.
6. Quesnell MM, Zhang Y, de Avila DM, Bertrand KP, Reeves JJ. Immunization of male mice with luteinizing hormone-releasing hormone
fusion proteins reduces testicular and accessory sex gland function.
Biol Repro 63:347–353, 2000.
7. Ladd A, Walfield A, Tsong YY, Thau R. Active immunization against
LHRH alone or combined with LHRH analogue treatment impedes
growth of androgen dependent prostatic carcinoma. Am J Reprod
Physiol 34:200–206, 1995.
8. Simms MS, Sholfield DP, Jacobs E, Michaeli D, Broome P, Humphreys JE, Bishop MC. Anti-GnRH antibodies can induce castrate
822
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
levels of testosterone in patients with advanced prostate cancer. Br J
Cancer 83:443–446, 2000.
Eng MH, Charles LG, Ross BD, Chrisp CE, Pienta KJ, Greenberg NM,
Hsu CX, Sanda MG. Early castration reduces prostatic carcinogenesis
in transgenic mice. Urology 54:1112–1119, 1999.
Raghow S, Kuliyev E, Steakley M, Greenberg N, Steiner MS. Efficacious chemoprevention of primary prostate cancer by flutamide in an
autochthonous transgenic model. Cancer Res 60:4093–4097, 2000.
Wechter WJ, Leipold DD, Murray D, Quiggle D, McCracken JD,
Barrios RS, Greenberg NM. E-7869 (R-flurbiprofen) inhibits progression of prostate cancer in the TRAMP mouse. Cancer Res 60:2203–
2208, 2000.
Fekete M, Redding TW, Comaru-Schally AM, Pontes JE, Connelly
RW, Srkalovic G, Schally AV. Receptors for luteinizing hormonereleasing hormone, somatostatin, prolactin, and epidermal growth factor in rat and human prostate cancers and in benign prostate hyperplasia. Prostate 14:191–208, 1989.
Emons G, Ortmann O, Schulz KD, Schally AV. Growth-inhibitory
actions of analogues of luteinizing hormone releasing hormone on
tumor cells. Trends in Endocrinology and Metab 8:355–361, 1997.
Jungwirth A, Schally AV, Pinski J, Halmos G, Groot K, Armatis P,
Vadillo-Buenfil M. Inhibition of in vivo proliferation of androgenindependent prostate cancers by an antagonist of growth hormonereleasing hormone. J Cancer 75:1585–1592, 1997.
Truett GE, Heeger P, Mynatt RL, Truett AA, Walker JA, Warman ML.
Peparation of PCR-quality mouse genomic DNA with hot sodium
hydroxide and Tris (HotSHOT). Biotechniques 29:52–54, 2000.
Hem A, Smith AJ, Solberg P. Saphenous vein puncture for blood
sampling of the mouse, rat, hamster, gerbil, guinea pig, ferret, and
mink. Lab Anim 32:364–368, 1998.
Sad S, Gupta HM, Talwar GP, Raghupathy R. Hyporesponsiveness to
a GnRH vaccine in a non-responder mouse strain is T-cell mediated. J
Reprod Immunol 20:189–194, 1991.
Han G, Foster BA, Mistry S, Buchanan G, Harris JM, Tilley WD,
Greenberg NM. Hormone status selects for spontaneous somatic androgen receptor variants that demonstrate specific ligand and cofactor
dependent activities in autochthonous prostate cancer. J Biol Chem
276:11204–11213, 2001.
Gregory CW, Johnson RT, Mohler JL, French FS, Wilson EM. Androgen receptor stabilization in recurrent prostate cancer is associated
with hypersensitivity to low androgen. Cancer Res 61:2892–2898,
2001.
Linja MJ, Savinainen KJ, Saramaki OR, Tammela TL, Vessella RL,
Visakorpi T. Amplification and overexpression of androgen receptor
gene in hormone refractory prostate cancer. Cancer Res 61:3550–
3555, 2001.
Srkalovic G, Bokser L, Radulovic S, Korkut E, Schally AV. Receptors
for luteinizing hormone releasing hormone (LHRH) in Dunning R3327
prostate cancers and rat anterior pituitaries after treatment with a sustained delivery system of LHRH antagonist SB-75. Endocrinology
127:3052–3060, 1990.
Halmos G, Arencibia JM, Schally AV, Davis R, Bostwick DG. High
incidence of receptors for luteinizing hormone releasing hormone
(LHRH) and LHRH receptor gene expression in human prostate cancers. J Urol 163:623–629, 2000.
IMMUNIZATION DOES NOT DECREASE PROSTATE CANCER